Recovery potential of cavitation-induced injured cells of common spore-forming bacteria in skim milk exposed to ultrasonication

The presence of spore-forming microorganisms and their adhesion to contact surfaces in dairy plants is a major concern because dairy products are prone to cross-contamination. Spore formers and their spores can survive milk processing treatments due to their heat resistance. One source of these spore formers is bacterial biofilms, which grow and accumulate on most surfaces in dairy industrial plants, such as pipes, heat exchangers, pasteurized plates, and storage tanks. Their endospores form biofilms by attaching to these surfaces and germinating when conditions become more favorable. The cross-contamination of dairy products by bacterial biofilms may lead to reduced shelf life and spoilage. To minimize the problem caused by thermoduric bacteria, the dairy industry adopts several approaches. Pasteurization is an irreplaceable technique for milk processing. Unfortunately, some bacteria and endospores are resistant to heat treatment, which can grow and cause spoilage of dairy products. Thus, alternative approaches that could help to reduce microbial counts are needed. In our previous study, we demonstrated the effectiveness of ultrasonication to inactivate spore formers and reduce the overall microbial counts in milk. In the current study, we investigated the recovery of cavitation-induced injured cells during the storage of ultrasonicated skim milk. Three common spore formers—Geobacillus stearothermophilus (ATCC 15952), Bacillus licheniformis (ATCC 6634), and Bacillus sporothermodurans (DSM 10599)—were selected to conduct challenge studies by inoculating skim milk samples and exposing them to ultrasonication (10 min each at 80% amplitude). This treatment was done in an ice bath to control the resultant temperature increase. The ultrasonicated skim milk samples were then held for 1, 2, 4, or 12 h in the refrigerator (4°C) to study the recovery of cells following cavitation-induced injury. Ultrasonication resulted in cell injury, as demonstrated by scanning electron microscopy. The injured cells can potentially recover under appropriate conditions during the storage of ultrasonicated milk and could affect the microbiological quality of milk and products manufactured with such milk. The respective bacterial counts for the 3 organisms in the spiked skim milk, on average, were approximately 6.0 log cfu/mL; immediately after ultrasonication, these counts decreased to 3.50 ± 0.02, 4.38 ± 0.02, and 3.75 ± 0.05 log cfu/mL for G. stearothermophilus, B. licheniformis, and B. sporothermodurans, respectively. During 12 h of subsequent incubation at 4°C, their counts increased to 4.17 ± 0.05, 5.25 ± 0.1, and 5.69 ± 0.06 log cfu/mL, respectively. All experiments were done in triplicate for all 3 bacteria. To conclude, slow recovery of injured cells of spore-forming bacteria is possible in ultrasonicated milk during storage under refrigeration conditions.